Cell-surface sialic acids are essential in mediating a variety of physiological and pathological processes. Sialic acid chemistry and biology remain challenging to investigate, demanding new tools for probing sialylation in living systems. The metabolic glycan labeling (MGL) strategy has emerged as an invaluable chemical biology tool that enables metabolic installation of useful functionalities into cell-surface sialoglycans by “hijacking” the sialic acid biosynthetic pathway. Here we review the principles of MGL and its applications in study and manipulation of sialic acid function, with an emphasis on recent advances.

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N-azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N-acetylglucosamine (GlcNAc) and incorporated into N-linked glycans, and possibly intracellular O-GlcNAc. Click-labeling with fluorescent probes enables visualization of newly synthesized N-linked glycans. N-glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N-glycosylation is regulated in a developmental stage-dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N-linked glycosylation in plants.

Molecular imaging of glycans has been actively pursued in animal systems for the past decades. However, visualization of plant glycans remains underdeveloped, despite that glycosylation is essential for the life cycle of plants. Metabolic glycan labeling in Arabidopsis thaliana by using N-azidoacetylglucosamine (GlcNAz) as the chemical reporter is reported. GlcNAz is metabolized through the salvage pathway of N-acetylglucosamine (GlcNAc) and incorporated into N-linked glycans, and possibly intracellular O-GlcNAc. Click-labeling with fluorescent probes enables visualization of newly synthesized N-linked glycans. N-glycosylation in the root tissue was discovered to possess distinct distribution patterns in different developmental zones, suggesting that N-glycosylation is regulated in a developmental stage-dependent manner. This work shows the utility of metabolic glycan labeling in elucidating the function of N-linked glycosylation in plants.

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium-mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N-(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell-surface glycans. This conversion chemically mimics the enzymatic de-N-acetylation of N-acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell-surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell-surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.

Thousands of intracellular proteins are post-translationally modified with O-GlcNAc, and O-GlcNAcylation impacts the function of modified proteins and mediates diverse biological processes. However, the ubiquity of this important glycosylation makes it highly challenging to probe the O-GlcNAcylation state of a specific protein at the cellular level. Herein, we report the development of a FLIM–FRET-based strategy, which exploits the spatial proximity of the O-GlcNAc moiety and the attaching protein, for protein-specific imaging of O-GlcNAcylation in single cells. We demonstrated this strategy by imaging the O-GlcNAcylation state of tau and β-catenin inside the cells. Furthermore, the changes in tau O-GlcNAcylation were monitored when the overall cellular O-GlcNAc was pharmacologically altered by using the OGT and OGA inhibitors. We envision that the FLIM–FRET strategy will be broadly applicable to probe the O-GlcNAcylation state of various proteins in the cells.

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor-associated glycans by using ligand-targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer-cell-specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue-specific manner. Subsequently, a copper-free click reaction was performed in vivo to chemoselectively conjugate the azides with a near-infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor-associated glycans by using ligand-targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer-cell-specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue-specific manner. Subsequently, a copper-free click reaction was performed in vivo to chemoselectively conjugate the azides with a near-infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.

Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging.

Alkynes can be metabolically incorporated into biomolecules including nucleic acids, proteins, lipids, and glycans. In addition to the clickable chemical reactivity, alkynes possess a unique Raman scattering within the Raman-silent region of a cell. Coupling this spectroscopic signature with Raman microscopy yields a new imaging modality beyond fluorescence and label-free microscopies. The bioorthogonal Raman imaging of various biomolecules tagged with an alkyne by a state-of-the-art Raman imaging technique, stimulated Raman scattering (SRS) microscopy, is reported. This imaging method affords non-invasiveness, high sensitivity, and molecular specificity and therefore should find broad applications in live-cell imaging.